Dihydrotachysterol: a Calcium Active Steroid Not Dependent Upon Kidney Metabolism

Dihydrotachysterol: a calcium active steroid not dependent upon kidney metabolism

Harold E. Harrison, Helen C. Harrison

J Clin Invest. 1972;51(7):1919-1922. https://doi.org/10.1172/JCI106996.

Concise Publication

The activity of dihydrotachysterol and cholecalciferol was determined in nephrectomized or sham-operated vitamin D- depleted rats using in vitro transport of calcium and phosphate by everted intestinal preparations as the index of physiologic response. The activity of dihydrotachysterol was not reduced by absence of the kidneys whereas that of cholecalciferol was markedly inhibited so that at least a 10-fold greater dose of the latter was necessary to produce an equivalent effect in the nephrectomized rat as in the control. Dihydrotachysterol is therefore equipotent with cholecalciferol in the anephric rat although much less active in the intact animal.

Find the latest version: https://jci.me/106996/pdf Dihydrotachysterol: a Calcium Active Steroid Not Dependent upon Kidney Metabolism

HARoLD E. HARRISON and HELEN C. HARRISON From the Departments of Pediatrics, Baltimore City Hospitals and Johns Hopkins University School of Medicine, Baltimore, Maryland 21224

A B S TR A C T The activity of dihydrotachysterol and body fluids. The pharmacologic effect requires amounts cholecalciferol was determined in nephrectomized or of vitamin D many fold that needed for physiologic sham-operated vitamin D-depleted rats using in vitro action and merges into the toxic action of vitamin D transport of calcium and phosphate by everted intestinal which results in hypercalcemia and soft tissue calcifi- preparations as the index of physiologic response. The cation. There is an apparent dissociation between these activity of dihydrotachysterol was not reduced by ab- physiologic and pharmacologic actions. Dihydrotachy- sence of the kidneys whereas that of cholecalciferol sterol2 is much less potent than the calciferols in the was markedly inhibited so that at least a 10-fold greater vitamin D-depleted animal but has actually greater phar- dose of the latter was necessary to produce an equiva- macologic potency than the calciferols in the hypopara- lent effect in the nephrectomized rat as in the control. thyroid subject (2). A possible clue to this discrepancy Dihydrotachysterol is therefore equipotent with chole- lies in the metabolic alterations which calciferol or di- calciferol in the anephric rat although much less active hydrotachysterol undergo in being transformed into in the intact animal. physiologically active compounds. Cholecalciferol is converted to a 25-hydroxy derivative in the liver (3) and then is further metabolized by INTRODUCTION the kidney to 1,25-dihydroxycholecalciferol (4). The 1, Several molecular forms of vitamin D or related acti- 25-dihydroxy derivative is probably the active compound vated sterols derived from the UV irradiation of 7- which is responsible for the physiologic effect of vita- dehydrocholesterol (cholecalciferol and dihydrotachy- min D. Dihydrotachysterol is hydroxylated in the 25- sterols) or of ergosterol (ergocalciferol and dihydro- position (5) but there is no evidence of a 1,25-di- tachysterol2) have both physiologic and pharmacologic hydroxy compound. If 25-hydroxydihydrotachysterol actions. The former are determined by the healing of were not a suitable substrate for the kidney mitochon- the skeletal lesions of rickets or by restoration to nor- drial system which catalyzes the 1-hydroxylation of 25- mal of the reduced serum calcium concentrations in hydroxycholecalciferol its relative lack of activity in the vitamin D-depleted animals or man. These physiologic physiologic assay would be explained. Since this meta- effects are closely correlated with the action of vitamin bolic step occurs primarily and possibly exclusively in D in increasing intestinal transport of calcium which the kidney (4) this thesis could be tested by comparing is thought to be its primary action (1). The pharma- the effect of nephrectomy on the activities of calciferol cologic effect is measured by increase of serum calcium and dihydrotachysterol in vitamin D-depleted rats. A concentrations in the hypoparathyroid animal (2) or response to 25-hydroxydihydrotachysterol by the in- patient and is associated with both an increase of intestine of vitamin D-deficient anephric rats was shown testinal transport of calcium and altered bone metabo- by Hallick and DeLuca (5). The data in this paper lism leading to solubilization of bone mineral and extend these studies and show clearly that the presence maintenance of higher concentrations of calcium ion in of the kidneys markedly increases the potency of cholecalciferol but does not affect that of These studies were presented in part at the meeting of the dihydrotachysterol. Federation of American Societies for Experimental Biology, METHODS 10 April 1972. Received for publication 28 February 1972 and in revised Weanling rats were depleted of vitamin D by a standard diet form 27 April 1972. (6). The completeness of vitamin D depletion was checked The Journal of Clinical Investigation Volume 51 July 1972 1919 by the reduction of serum calcium concentrations after 3 wk TABLE I IF of diet. At this time, the rats were shifted to a low protein Comparison of Effects of Cholecalciferol (CC) and Dihydro- (8% egg albumin), vitamin D-free diet which was also tachysterol (DHT) on In Vitro Calcium and Phosphate very low in potassium, sodium, and phosphate. On this diet vitamin D-depleted rats tolerated nephrectomy, continued to Transport by Everted Intestinal Loops of Sham- feed, and continued in good physiologic condition for several Operated and Nephrectomized Vitamin days after nephrectomy making possible an accurate mea- D-Depleted Rats sure of vitamin D action on the intestinal transport of calcium and phosphate. After 3 days of this diet, animals were DHT CC either bilaterally nephrectomized or subjected to sham operation as controls. 3-4 hr postoperatively the rats were Steroid Sham Nephrectomized Sham Nephrectomized given by stomach tube varying amounts of either crystalline cholecalciferol (ICN Nutritional Biochemicals Div., Cleve- Calcium transport-duodenum (Cs/Cm) land, Ohio) or crystalline dihydrotachysterol2 (Philips- 0 2.02 ±0. 16 1.874-0.11 2.02 10.16 1.87 40.I 1 duPhar, Amsterdam) dissolved in propylene glycol. Dihy- (13) (11) (13) (11) drotachysterol2 was used because it is the compound which $ * ** ** has been given to patients with renal insufficiency. Untreated 2.5 2.8640.18 3.57±0.23 8.57±0.32 He 3.04±0.34 animals were given only solvent. 42 hr later, the animals (6) (7) (7) (7) were bled under pentobarbital anesthesia and the entire 10 3.00:0.34 4.2940.69 9.29±1.26 ** 3.99:40.82 small intestine removed. The assay for vitamin D effect was (6) (7) (9) (7) the enhancement of calcium transport and of phosphate 25 7.491:1.30 11.67±2.01 11.0 ±0.99 ** 6.20±-0.89 transport by everted intestinal loops in vitro as has been (6) (6) (8) (7) described (7). Calcium transport was measured in duodenal Calcium transport-ileum (Cs/Cm) and ileal segments while phosphate transport was deter- 0 0.89±0.04 * 0.71410.02 0.89 10.04 * 0.714:0.02 mined in mid-intestinal segments. The buffer for calcium (13) (12) (13) (12) transport studies contained sodium in a concentration of 50 mEq/liter since this has been found to give maximal calcium 2.5 1.04±0.07 1.48410.19 2.6910.27 ** 0.971:0.04 transport in both duodenum and ileum as well as in colon (7) (7) (5) (7) 10 1.5540.27 1.56±0.16 3.1740.33 ** 0.9740.08 (8). The measure of solute transport was the final ratio (6) (8) (9) (8) of concentration of solute in serosal and mucosal fluids 25 2.1140.18 2.51 40.20 2.8940.34 ** 1.7840.20 (Cs/Cm), the initial ratio being unity. (6) (6) (6) (7) RESULTS Phosphate transport (Cs/Cm) 0 1.54±40.07 1.62 40.14 1.54±40.07 1.62 40.14 The data tabulated in Table I indicate the effects of (13) (12) (13) (12) cholecalciferol and dihydrotachysterol2 treatment on 2.5 1.68 ±0.08 1.78±+0.15 2.69 ±0.19 ** 1.55 ±0.06 calcium and phosphate transport in sham-operated and (6) (7) (7) (7) nephrectomized rats. The calcium transport in duodenum 10 2.02±0.16 2.03±0.09 3.45±0.91 ** 1.6140.14 (6) (8) (9) (7) and ileum and the phosphate transport in mid-intestine 25 2.61 ±0.35 2.9440.38 3.20±0.29 * 2.01 ±0.15 roughly parallel each other and there is no evidence (6) (6) (8) (7) of a major differential effect of type of steroid or of kidney removal on intestinal site or on the ion trans- Values are means 5sEM. No. of animals in group in parentheses. * Difference between values statistically significant P < 0.05. ported. 2.5 jig of cholecalciferol produces a near maxi- ** Difference between values highly significant P < 0.01. mal enhancement of calcium and phosphate transport in intestine from sham-operated animals whereas di- hydrotachysterola has very little effect at this dose. This denum although there is a decrease in ileal transport. amount of cholecalciferol has only a slight action on When 2.5 ug of cholecalciferol are given 18 hr before calcium transport and no effect on phosphate transport, nephrectomy and intestinal transport determined 42 hr however, in intestine from nephrectomized rats. With postnephrectomy Cs/Cm ratios for calcium transport increasing dosage of both compounds it becomes ap- in duodenum and ileum are 7.86+0.37 and 2.26±0.23 parent that the absence of kidney tissue does not impair and that for phosphate 2.49±0.28. The effect of uremia dihydrotachysterol effect whereas cholecalciferol action is thus not sufficient to inhibit cholecalciferol effect is markedly inhibited so that in the nephrectomized rat once conversion to the active metabolite by the kidney dihydrotachysterol is as, or more potent than com- has occurred. Sham-operated rats pretreated with 2.5 parable doses of cholecalciferol. lig of cholecalciferol do show a greater increment of In these experiments the uremic state per se appar- transport than do the nephrectomized rats but this is not ently has little effect on the intestinal transport sys- necessarily evidence of an inhibitory effect of uremia tems apart from metabolic conversion of vitamin D and may indicate that conversion of cholecalciferol to since, in the untreated vitamin D-depleted rat, neph- 1,25-dihydroxycholecalciferol continues beyond the 18 rectomy causes no significant reduction in the already hr interval between administration and operation so low level of in vitro transport of calcium in the duo- that the sham-operated rats have a greater response 1920 H. E. Harrison and H. C. Harrison due to continued formation and therefore greater con- bone by this steroid in the nephrectomized animal. A centration of the active metabolite. greater physiologic effect of dihydrotachysterol than In Table II are summarized serum calcium, phos- of cholecalciferol in the nephrectomized rat is also phate, and citrate concentrations of the animals from suggested by results of the calcium transport studies which the data of Table I were obtained. When the (Table I). vitamin D-depleted rats are changed from the original diet containing 0.6% Ca and 0.5% P to the low protein, DISCUSSION phosphate-free diet without change in calcium intake The data show that a major difference between chole- the serum calcium rises to normal as the serum in- calciferol and dihydrotachysterol is activation of the organic phosphate drops. The low phosphate intake former by the kidney presumably by transformation to prevents any marked increase of serum phosphate after a highly active compound, 1,25-dihydroxycholecalciferol, nephrectomy. In the anephric rats there is a marked whereas dihydrotachysterol activity is not increased by rise in concentrations of serum citrate and of calcium kidney. Under the conditions of this study, the effects which has been observed by others (9). This probably of uremia on the calcium and phosphate transport sys- is the result of the removal of the citrate metabolizing tems of the intestine are not a major factor, and there tissue of the kidney. The rise of citrate and of calcium is certainly no reduction of dihydrotachysterol effect in concentrations is greater in rats receiving 10 and 25 i'g the anephric rat. of dihydrotachysterol than in those given comparable Inasmuch as dihydrotachysterol in large doses does amounts of cholecalciferol. This may indicate relatively enhance calcium and phosphate transport by intestine greater mobilization of citrate and of calcium from of vitamin D-deficient rats and cholecalciferol in simi- lar doses also does the same in preparations from rats the kidney is not completely essen- TABLE II nephrectomized Concentrations of Calcium, Phosphate, and Citrate in Serum of tial for vitamin D effect even though it metabolizes Nephrectomized or Sham-Operated Vitamin D-Depleted Rats calciferol to a much more active compound. This may Given Varying Doses of Cholecalciferol (CC) or mean that derivatives other than the 1,25-dihydroxy Dihydrotachysterol (DHT) 4 hr after Operation. compounds have an action on intestinal transport of Blood Taken 42 hr after Operation calcium. Since both calciferol and dihydrotachysterol are metabolized to 25-hydroxy compounds these may be DHT CC physiologically active but with a much lower potency than the 1,25-dihydroxy calciferol, or alternately other Steroid Sham Nephrectomized Sham Nephrectomized dihydroxy compounds such as the 21,25 dihydroxy- ;6g cholecalciferol isolated by Suda et al. (10) may be Calcium, mg/100 ml 0 10.08140.06 12.93140.30 10.08 4±0.06 12.93 40.30 formed in the absence of the kidney and be somewhat (12) (12) (10) (12) active in the intestinal assay. It is also possible that 2.5 10.1340.11 13.9740.30 11.90-0.19 13.03±0.31 at the 1 carbon could occur in tissues (7) (7) (7) (7) hydroxylation 10 10.37 ±0.15 13.93 ±0.43 12.29 ±0.24 12.89±i0.37 other than the kidney but at a much lower rate. If so, (6) (8) (9) (8) such extrarenal metabolism must be as great or greater 25 11.65±0.27 17.20±0.94 12.84±0.21 13.8440.71 (6) (6) (5) (7) for dihydrotachysterol than for cholecalciferol and this seems unlikely. In either instance, 25-hydroxydihydro- Citrate, mg/100 ml 0 4.86 40.33 18.03 ± 1.96 4.86 40.33 18.03 ± 1.96 tachysterol is not metabolized to a more active com- (6) (6) (6) (6) pound by the kidney presumably because it is not a suit- 2.5 4.51±40.31 14.82 43.02 - 15.5442.22 able substrate for the kidney 1-hydroxylation system. (5) (7) (4) 10 5.63 ±0.52 22.33 43.82 7.46 ±0.26 12.03±1.13 This can explain the much lower potency of dihydro- (4) (6) (8) (7) tachysterol than of calciferol in the vitamin D-depleted 25 5.33±0.13 31.5044.41 - 21.33±1.75 animal. Since at pharmacologic doses dihydrotachy- (5) (5) (3) sterol is more potent than calciferol in the hypopara- Phosphate, mg/100 ml thyroid subject (2), dihydrotachysterol or its deriva- 0 1.20±0.10 2.32±0.36 1.20±0.10 2.32±0.36 (I l) (12) (I1) (12) tive is evidently more active than the calciferol metabo- 2.5 1.5440.17 2.12±0.21 2.52±0.29 2.27±0.23 lite with respect to solubilization of bone mineral in (7) (7) (6) (7) the absence of parathyroid hormone. 10 1..13±0.15 2.74±0.17 2.29±0.24 2.06±0.17 (6) (8) (9) (8) In patients with renal disease, calcium malabsorption 25 1.71±0.16 2.98±0.40 3.00±0.35 2.25±0.21 refractory to ordinary amounts of vitamin D is a well (6) (6) (5) (4) recognized problem (11). Lack of the formation of Values are means ASEM. No. of animals in group in parentheses. 1,25-dihydroxycalciferol due to severe reduction of ac- Dihydrotachysterol and Kidney 1921 tive renal metabolic tissue has been postulated to be the 25-hydroxycholecalciferol. A biologically active metabo- mechanism. Under these conditions dihydrotachysterol lite of vitamin Ds. Biochemistry. 7: 3317. 4. Fraser, D. R., and E. Kodicek. 1970. Unique biosyn- may be the preferable steroid for treatment rather than thesis by kidney of a biologically active vitamin D larger doses of calciferol since the amount of dihydro-_ metabolite. Nature (Lond.) 228: 764. tachysterol required will not vary with changes in 5. Hallick, R. B., and H. F. DeLuca. 1972. Metabolites amount of functioning kidney tissue. The dosage of of dihydrotachysterol, in target tissues. J. Biol. Chem. more 247: 91. dihydrotachysterol should be uniform, therefore, 6. Harrison, H. C., H. E. Harrison, and E. A. Park. and not greatly different from that necessary to produce 1958. Vitamin D and citrate metabolism. Effect of vita- a physiologic action on calcium absorption in the sub- min D in rats fed diets adequate in both calcium and ject with intact kidneys. Liu and Chu (12) and Kaye, phosphorus. Am. J. Physiol. 192: 432. Chatterjee, Cohen, and Sagar (13) have demonstrated 7. Harrison, H. E., and H. C. Harrison. 1960. Transfer of Ca'5 across the intestinal wall in vitro in relation to that dihydrotachysterol does increase calcium absorp- the action of vitamin D and cortisol. Am. J. Physiol. tion by the patient with renal insufficiency and sup- 199: 265. presses secondary hyperparathyroidism more effectively 8. Harrison, H. C., and H. E. Harrison. 1969. Calcium than equivalent doses of calciferol. transport by rat colon in vitro. Am. J. Physiol. 217: 121. 9. Freeman, S., and T. S. Chang. 1950. Role of the kidney ACKNOWLEDGMENTS and of citric acid in production of a transient hypercalcemia following nephrectomy. Am. J. Physiol. 160: We appreciate the skillful technical assistance of Mrs. 355. Evelyn Bull. 10. Suda, T., H. F. DeLuca, H. K. Schnoes, G. Ponchon, This investigation was supported by U. S. Public Health V. Tanaka, and M. F. Holick. 1970. 21,25-dihydroxy- Service, National Institutes of Health Grant AM-00668. cholecalciferol. A metabolite of vitamin D3 preferen- tially active on bone. Biochemistry. 9: 2917. REFERENCES 11. Lumb, G. A., E. B. Mawer, and S. W. Stanbury. 1971. The apparent vitamin D resistance of chronic 1. DeLuca, H. F. 1967. Mechanism of action and meta- renal failure. A study of the physiology of vitamin D bolic fate of vitamin D. Vitam. Horm. 28: 315. in man. Am. J. Med. 50: 421. 2. Harrison, H. E., H. C. Harrison, and F. Lifshitz. 1968. 12. Liu, S. H.. and H. I. Chu. 1943. Studies of calcium Interrelation of vitamin D and parathyroid hormone. and phosphorus metabolism with special reference to The responses of vitamin D depleted and of thyropara- pathogenesis and effects of dihydrotachysterol (A. T. thyroidectomized rats to ergocalciferol and dihydrotachy- 10) and iron. Medicine. 22: 103. sterol. In Parathyroid Hormone and Thyrocalcitonin 13. Kaye, M., M. B. Chatterjee, G. F. Cohen, and S. (Calcitonin). R. V. Talmage and L. F. Belanger, edi- Sagar. 1970. Arrest of hyperparathyroid bone disease tors. Excerpta Medica Foundation, Amsterdam. 455. with dihydrotachysterol in patients undergoing chronic 3. Blunt, J. W., H. F. DeLuca, and H. R. Schnoes. 1968. dialysis. Ann. Intern. Med. 73: 225.

1922 H. E. Harrison and H. C. Harrison